P α syn

Free-floating sections (30 μm) were incubated in 0.1 M PBS containing 5% normal donkey serum and 0.3% Triton X-100 for 1 h, and subsequently incubated overnight with specific primary antibodies (Tuj-1, 1:1000, BioLegend, San Diego, CA, USA; α-syn, 1:1000, BD Bioscience, USA; p-α-syn, 1:1000, Abcam, Cambridge, UK, Amyloid beta, Aβ, 1:1000, BioLegend (6E10), San Diego, CA, USA; A11, 1:1000, Invitrogen, Waltham, MA USA; NOX4 (1:500, Santa Cruz Biotechnology, USA) in 2% normal donkey serum (Vector Laboratories, Burlingame, CA, USA) in PBS at 4 and incubated with a 1:200 dilution of Alexa Fluor-conjugated donkey anti-rabbit (488) or donkey anti-mouse (546) antibodies (Invitrogen, Grand Island, NY, USA) for 1 h at room temperature and mounted on glass slides using Vectashield (Vector Laboratories, Burlingame, CA, USA). Fluorescent signals were evaluated on a confocal microscope (LSM 710, Carl Zeiss, Oberkochen, Germany) [29 (link)].

Mouse hippocampus protein lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently immunoblotted onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk at room temperature for 1 h. After TBST washing (three times, 5 min per wash), the membranes were incubated with the indicated primary antibodies at 4 °C overnight with shaking. The primary antibodies included β-actin (Sigma-Aldrich, A5316), lamin B1 (HUABIO Antibodies, ET1606-27), HIF-1α (Abcam, ab228649), HIF-2α (Novus, NB100-122), Acer2 (PA5-39016), PP2A (Invitrogen, MA5-32920), p-PP2A (Invitrogen, MA5-32158), α-syn (BD, 8249518), and p-α-syn (Abcam, ab51253). After incubation, the membranes were washed three times and then incubated at room temperature for 1 h with secondary antibodies, including IRDye 680RD goat anti-mouse IgG (H + L) (Licor, 926-68070), IRDye 680RD goat anti-rabbit IgG (H + L) (Licor, 926-68071), IRDye 800CW goat anti-mouse IgG (H + L) (Licor, 926-32210), IRDye 800CW goat anti-rabbit IgG (H + L) (Licor, 926-32211). Membranes were scanned using a detection system (Odyssey, USA), and band intensities were normalized to β-actin. Statistical analyses were performed using ImageJ and GraphPad software.